We have isolated a large collection of temperature-sensitive (ts) mutants in established Chinese Hamster cell lines (CHO, V79) and initiated their characterization. Approximately 5% are phenotypically ts DNA-. These will be further tested for the effect of the ts cell function for adenovirus DNA synthesis in vivo and in vitro so as to define biochemically the defect. A previously isolated class of ts DNA- mutants in mouse 3T3 has been shown to be restrictive for polyomaviral DS in vivo and in vitro. A biochemical defect has been identified in DNA polymerase (polAlpha2). These mutants and selected CHO mutants will be further utilized for isolation of the wild-type homolog of the mutant gene (by gene transfer and recombinant DNA methodologies) and subsequent definition of the mutant polypeptide. Finally, we will determine whether SV40 DNA synthesis can be positively effected in CHO by increasing levels of viral T antigen, viral origins of DNA replication, or both. If successful, such manipulations will be used to extend the utility of the CHO mutants to virus-cell interactions with SV40 as well, permitting the definition of additional cellular functions. Together, these studies should significantly increase our information on viral and cell DNA replication, a fundamental aspect of carcinogenesis.